Lang Lukas, Reinert Philipp, Diaz Cedric, Deponte Marcel
Faculty of Chemistry, Comparative Biochemistry, RPTU Kaiserslautern, D-67663, Kaiserslautern, Germany.
Faculty of Chemistry, Comparative Biochemistry, RPTU Kaiserslautern, D-67663, Kaiserslautern, Germany.
Redox Biol. 2024 Dec;78:103410. doi: 10.1016/j.redox.2024.103410. Epub 2024 Oct 24.
Class I glutaredoxins reversibly reduce glutathione- and nonglutathione disulfides with the help of reduced glutathione (GSH) using either a monothiol mechanism or a dithiol mechanism. The monothiol mechanism exclusively involves a single glutathionylated active-site cysteinyl residue, whereas the dithiol mechanism requires the additional formation of an intramolecular disulfide bond between the active-site cysteinyl residue and a resolving cysteinyl residue. While the oxidation of glutaredoxins by glutathione disulfide substrates has been extensively characterized, the enzyme-substrate interactions for the reduction of S-glutathionylated glutaredoxins or intramolecular glutaredoxin disulfides are still poorly characterized. Here we compared the thiol-specificity for the reduction of S-glutathionylated glutaredoxins and the intramolecular glutaredoxin disulfide. We show that S-glutathionylated glutaredoxins rapidly react with a plethora of thiols and that the 2nd glutathione-interaction site of class I glutaredoxins lacks specificity for GSH as a reducing agent. In contrast, the slower reduction of the partially strained intramolecular glutaredoxin disulfide involves specific interactions with both carboxylate groups of GSH at the 1st glutathione-interaction site. Thus, the dithiol mechanism of class I glutaredoxins promotes specificity for GSH as a reducing agent, which might explain the prevalence of dithiol glutaredoxins in pro- and eukaryotes.
I类谷氧还蛋白借助还原型谷胱甘肽（GSH），通过单硫醇机制或二硫醇机制可逆地还原谷胱甘肽二硫化物和非谷胱甘肽二硫化物。单硫醇机制仅涉及单个谷胱甘肽化的活性位点半胱氨酰残基，而二硫醇机制则需要在活性位点半胱氨酰残基和一个解离半胱氨酰残基之间额外形成分子内二硫键。虽然谷胱甘肽二硫化物底物对谷氧还蛋白的氧化已得到广泛表征，但对于S-谷胱甘肽化谷氧还蛋白或分子内谷氧还蛋白二硫化物还原的酶-底物相互作用仍知之甚少。在此，我们比较了还原S-谷胱甘肽化谷氧还蛋白和分子内谷氧还蛋白二硫化物的硫醇特异性。我们发现，S-谷胱甘肽化谷氧还蛋白能与大量硫醇迅速反应，且I类谷氧还蛋白的第二个谷胱甘肽相互作用位点对作为还原剂的GSH缺乏特异性。相比之下，部分张力的分子内谷氧还蛋白二硫化物的较慢还原涉及在第一个谷胱甘肽相互作用位点与GSH的两个羧基的特异性相互作用。因此，I类谷氧还蛋白的二硫醇机制促进了对作为还原剂的GSH的特异性，这可能解释了二硫醇谷氧还蛋白在原核生物和真核生物中的普遍存在。
